Compliant dehyrated tissue for implantation and process of making the same

ABSTRACT

A process for preparing pliable soft tissue specimen which are resistant to cracking and devoid of viable cells includes the steps of treating native soft tissue obtained from a donor by a gradually increasing gradient of aliphatic alcohol or other suitable water miscible polar organic solvent until the last alcohol (or other solvent) solution has at least 25% by volume of the organic liquid. Thereafter, the tissue specimen is treated with a solution containing glycerol or low molecular weight (&lt;1000 D) polyethylene glycol, and polyethylene glycol of a molecular weight between approximately 6,000 to 15,000 D and heparin. Thereafter, the tissue specimen is briefly immersed in aqueous heparin solution, frozen and lyophilized. The tissue specimen is suitable for implantation as a homograft or xenograft, with or without rehydration.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is in the field of implant materials. Moreparticularly, the present invention is directed to compliant dehydratedimplant materials which have no viable cells, and can be stored andtransported without being immersed in liquid. The present invention isalso directed to the process of producing said implant materials.

2. Brief Description of the Prior Art

The use of autografts, homografts and xenografts for augmenting orreplacing defective tissues in humans and animals has been known for along time. From the standpoint of providing suitable materials forimplantation, augmenting or replacing hard tissues, such as bone,presents a different type of problem than augmenting or replacing softtissues. In the selection of substitute materials for hard tissue graft,the strength and hardness of the graft are important whereas complianceand flexibility are, generally speaking, less crucial.

On the other hand, in the selection of soft tissue materials forimplantation, compliance and flexibility of the graft material areusually of utmost importance because the soft tissue replacementmaterial usually must closely match the healthy functional tissue thatwill be replaced. In this regard it must be remembered that natural softtissue containing collagen is strong and able to withstand repeatedthree-dimensional stress as well as bending and deformation. Oftennatural soft tissue acts as a physical barrier that must maintain itsstructural integrity. Ideally, replacement or augmentation soft tissuethat is utilized in implantation should have the same characteristics asthe natural soft tissue that it replaces, and should be easy to obtain,store and transport. These, however are difficult goals that the priorart has been striving to attain, and up to the present invention onlywith moderate success.

More particularly, in accordance with one major approach in the priorart to preserve soft tissue for eventual implantation, tissues of humanor animal origins have been treated with chemicalmodifiers/preservatives, such as glutaraldehyde, which cross-linkscollagen and other proteins. The glutaraldehyde treated tissues havebeen shown to be adequately resistant to mechanical fatigue as well asbiodegradation when implanted in human patients. However, theglutaraldehyde cross-linking alters the viscoelastic properties oftissues, and therefore, as a result of host response undesirablecalcification and build-up of peripheral granulation tissues usuallyoccur in the implants with time. Glutaraldehyde is an effective biocidal(sterilyzing) agent, but when exposed to air it slowly loses itsbiocidal effectiveness. Therefore, the tissue intended for implantation(bioprosthesis) must be kept in glutaraldehyde solution during storageand transportation and the package including the glutaraldehyde soakedbioprosthesis must be kept tightly sealed. Moreover, it must not beexposed to significantly elevated temperature. Because of theserequirements the costs of utilizing glutaraldehyde-treated soft tissuebioprostheses are high. Glutaraldehyde is toxic, and therefore it mustbe carefully removed from the bioprosthesis by rinsing beforeimplantation. This represents still another disadvantage ofglutaraldehyde-treated bioprostheses.

Another major approach for providing soft tissue bioprosthesis in theprior art utilizes liquid sterilants other than glutaraldehyde. Some ofthese alternative approaches also avoid the calcification problemsassociated with glutaraldehyde treated implants. However, in accordancewith these processes also, to avoid brittleness and to more-or-lessmaintain the physical integrity of the bioprostheses the tissues have tobe maintained, stored and transported in liquid media up to the timeimmediately preceding implantation.

Still another alternative method for providing soft tissue bioprosthesesis the use of cryo-preserved fresh tissues of homograft (tissue from thesame species). Because of recent advances in cryo-preservation, thecryo-preserved fresh tissues have recently made homograft implantsrelatively more successful and more accepted as an alternative toglutaraldehyde-preserved xenograft. A serious disadvantage ofcryo-preserved bioprostheses is the difficulty to assure that they arefree of infectious disease agents. The costs of preparing and handlingof cryo-preserved bioprosthesis tissues is also very high because of theneed for keeping the tissues at all times below the usual or normalfreezer temperatures.

From among the numerous patent disclosures in the prior art directed topreparing and/or preserving biological tissue for implantation or otheruse as replacement tissue, U.S. Pat. Nos. 5,116,552 (Morita et al.) and5,336,616 (Livesey et al.) are mentioned as of interest to the presentinvention. U.S. Pat. No. 5,116,552 (Morita et al.) describes a processfor preparing lyophilized collagen sponge for medical applications, suchas artificial skin. The process of this reference comprises the steps ofimpregnating cross-linked collagen sponge with an aqueous solution of ahydrophilic organic solvent, freezing the sponge and thereafter vacuumdrying (lyophilizing) it. However, the resulting freeze-dried product isnot pliable and is not protected from cracking because the water and thehydrophilic solvent or solvents have been removed in the lyophilizationstep. U.S. Pat. No. 5,336,616 (Livesey et al.) describes treatment ofsoft tissue obtained from a source, such as a cadaver, with solutionscontaining antioxidants, protease inhibitors and antibiotics(stabilizing solution), with enzymes and detergents to remove viableantigenic cells (processing solution), and after decellularization witha cryopreservative solution that prevents destructive ice crystalformation while the tissue is frozen. The cryo-preserving solution mayinclude a combination of organic solvent and water. After lyophilizationthe product is stored and transported in a sealed container in an inertgas atmosphere, thus protected from atmospheric moisture. Prior toimplantation the tissue is rehydrated and must be restored withimmunotolerable viable cells to produce a permanently acceptable graftfor implantation.

Still other disclosures pertaining to the preparation and/orpreservation of biological tissue for implantation, or related subjects,can be found in U.S. Pat. Nos. 2,106,261; 2,610,625; 2,645,618;3,939,260; 4,277,238; 4,280,954; 4,300,243; 4,383,832; 4,578,067;4,703,108; 4,704,131; 4,760,131; 4,801,299; 4,911,915; 5,028,597;5,131,850; 5,674,290 and U.K. Patent Specification 716,161.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a soft tissue graftsuitable for implantation in humans or other mammals which graft afterrehydration has substantially the same mechanical properties as thenatural soft tissue from which the graft was obtained.

It is another object of the present invention to provide a soft tissuegraft that satisfies the foregoing objective, that is also devoid ofviable cells and does not require inoculation with viable cells prior toimplantation.

It is still another object of the present invention to provide a softtissue graft that satisfies the foregoing objectives, that can be storedand transported in a dehydrated form.

The foregoing and other objects and advantages are attained by a softtissue preparation that in its dehydrated state is compliant, resistscracking, is devoid of viable cells and which is obtained bysuccessively treating natural soft tissue:

with liquid compositions of gradually increasing concentrations of aC₁-C₃ alcohol, or other polar water miscible organic solvent in water,until the last of said liquid compositions contains at leastapproximately 25% by volume alcohol, or the other organic solvent, ormixtures thereof, the balance being water;

thereafter with a second liquid composition of aqueous glycerol or oflow molecular weight (<1000 D) polyethylene glycol, containing theglycerol or the low molecular weight polyethylene glycol, or mixturesthereof, in a concentration range of approximately 10 to 50% by volume,said second liquid composition also containing approximately 3-20%weight by volume polyethylene glycol of a molecular weight in the rangeof 6,000 D to 15,000 D and approximately 2 to 75 unit per milliliterheparin of a molecular weight greater than approximately 3 KD;

thereafter draining excess liquid from the soft tissue so treated;

thereafter immersing the soft tissue in an aqueous heparin solution ofapproximately 20 to 500 unit per milliliter concentration, and

thereafter freezing the tissue and lyophilizing the tissue to dryness.

The features of the present invention can be best understood togetherwith further objects and advantages by reference to the followingdetailed description of specific examples and embodiments.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following specification sets forth the preferred embodiments of thepresent invention. The embodiments of the invention disclosed herein arethe best modes contemplated by the inventors for carrying out theirinvention, although it should be understood that various modificationscan be accomplished within the parameters of the present invention.

In accordance with the present invention soft tissue intended for graftin mammals, including humans, is first obtained from a source, such ascadavers. Bovine, ovine, porcine tissue and soft tissue obtained fromother animals, such as sheep, serve as examples. Human soft tissue mayalso be used. Homografts, that is tissues implanted in the same speciesas the donor, as well as xenografts, that is tissues implanted inspecies different from the donor, can be prepared in accordance with thepresent invention. The types of tissues used in accordance with thepresent invention are generally the same which are normally used instate-of-the-art surgical procedures involving implantations of softtissues, primarily in humans. Examples of tissues frequently utilized inthese procedures are pericardium, aortic and pulmonary roots, tendons,ligaments, skin, peritonium, pleura, mitral and tricuspid valves.

The soft tissue excised from the donor is usually trimmed to removeloose excess or unneeded tissue and fat. Usually the tissue is then keptin saline solution. Thereafter, and in accordance with the presentinvention, the tissue is treated in a first aqueous solution containinga C₁-C₃ alcohol in relatively low concentration (approximately 15-35),and thereafter in a second aqueous solution of greater alcoholconcentration, in the range of approximately 25 to 75% volume by volume.(All concentrations described in this application are volume by volume,unless specifically stated otherwise.) The purpose of the treatment ofthe tissue specimen with the first and second solutions is to graduallyreplace the water content of the specimen with alcohol. Methyl, ethyland iso-propyl alcohols can be used for this purpose with ethyl alcoholbeing preferred. Other, non-toxic polar and water miscible organicsolvents e.g. acetonitrile, acetone or methyl-ethyl ketone can also beused instead of the above-listed alcohols, and mixtures of alcohols andorganic solvents are also suitable for use in the invention. Preferably,the first solution contains approximately 25% ethyl alcohol, the balancebeing water, and the second solution contains approximately 50% ethylalcohol, the balance being water.

Those skilled in the art will readily recognize that the foregoingmanipulations represent treatment of the tissue specimen with a stepwiseincreasing gradient of alcohol (or other suitable non-toxic watermiscible organic solvent) concentration, until a concentration of atleast approximately 25%, preferably approximately 50%, and at mostapproximately 75% alcohol (or other suitable solvent) concentration isreached. Instead of treating the tissue specimen with the aforesaidconcentration gradient in two steps, the specimen could also be treatedwith the gradient in three or more steps, or even with a continuouslyincreasing gradient until the upper limit of the alcohol (or othersuitable solvent) concentration is reached. The treatment with theincreasing gradient of alcohol (or other suitable solvent) concentrationis conducted at ambient temperature and is best performed by immersingthe tissue specimen in the solutions. The timing of the exposure of thetissue specimen to these solutions is not critical and is somewhatdependent on the thickness of the specimen. However sufficient time mustbe given for the solution to penetrate the specimen. Typically, 30minutes are sufficient and in the preferred embodiments of the processof the invention the tissue specimen are kept for approximately 30minutes in each of the first and second alcohol solutions.

After immersion (treatment) in the above-described alcohol solutions,the tissue specimen is immersed (treated) in a third solution thatcontains approximately 10 to 50% glycerol, approximately 3 to 20% weightby volume polyethylene glycol of a molecular weight in the range of6,000 D to 15,000 D and approximately 2 to 75 unit per milliliterheparin of a molecular weight greater than approximately 3 KD.Preferably, the third solution contains approximately 20% glycerol,approximately 5% (weight by volume) polyethylene glycol that has amolecular weight of approximately 8,000 D and approximately 50 unit permilliliter heparin. Instead of glycerol, a low molecular weight (<1000D) polyethylene glycol can be included in the third solution. Theduration of immersion in the third solution is also not critical,approximately 30 minutes are sufficient for very thin tissues such asovine, porcine, bovine or human pericardium, but for thicker tissueslonger times of exposure, such as 6 hours, or preferably 12 hours areconvenient and preferred.

After treatment with the third solution, the tissue specimen is removedtherefrom and excess liquid is allowed to drain from the specimen. Thespecimen is then briefly (for seconds as in a quick dip) immersed in, oris otherwise treated with aqueous heparin solution of approximately 20to 500 unit/ml concentration, and preferably of approximately 250ml/unit concentration, then the heparin solution is allowed to drainoff. Thereafter, the specimen is frozen in a manner usual in the art forfreezing specimens prior to lyophilization. Those skilled in the artunderstand that freezing is usually conducted in a freezer of ultra-lowtemperature, that is between approximately −60° C.-−80° C. Afterfreezing, the tissue specimen is lyophilized (dried in vacuo) in amanner known in the art.

Tissue samples processed in accordance with the invention tend to betranslucent and have a slight yellowish tint in color. Unlike tissueslyophilized from 100% water or physiological saline solution, thetissues of the invention are pliable, compliant and do not crack orbreak as a result of physical manipulations.

For use in surgical procedures as an implant, and for most testsconducted in accordance with the present invention to compare thetreated tissues with fresh tissues, the lyophilized tissues are firstrehydrated in physiological buffered saline. This is done by treating,preferably by immersing, the lyophilized tissue of the invention inphysiological buffered saline solution for approximately 5 minutes toone hour. The rehydrated tissues of the invention have an appearancethat is practically indistinguishable from the appearance of the freshtissue. Rehydration is typically conducted at ambient temperature. Itcan be done, other than in saline, in the patient's own blood, in tissueculture medium, and in low percentage (<10%) ethyl alcohol solution. Apreferred method of rehydrating tissue specimen in accordance with thepresent invention is in buffered saline of pH 7.4.

As noted above, except for testing the tissue specimen of the presentinvention, rehydration is performed only prior to use of the tissuespecimen for implantation. Otherwise the specimen are stored andtransported at ambient temperature in a sealed container protected fromatmospheric moisture. The lyophilized tissues can be readily sterilizedby gas phase sterilization methods, and can also be implanted withoutfirst being rehydrated.

The tissue specimen of the invention do not contain viable cells, buttests described below demonstrated that after rehydration the tissuespecimen are not cytotoxic and are compatible for host endothelial cellsto attach and proliferate on them. This attachment and proliferation ofhost cells and lack of cytotoxicity are important for long term survivalof most implants. The tissues of the invention are hemocompatible andresistant to platelet aggregation and thrombus formation. Tests,described below, also demonstrated that the collagen fibers of thenative tissue have remained substantially intact during the steps of theprocess of the invention, and are substantially intact in the rehydratedtissue.

Specific Examples and Description of Tests

(a) Preparation of Lyophilized Bovine or Ovine Pericardium

Fresh bovine and ovine pericardium was cut into strips and squares weredissected to remove loose tissues and fat. The tissues were immediatelyplaced in aqueous 25% ethyl alcohol solution for 30 minutes. The aqueous25% ethyl alcohol solution was replaced by aqueous 50% ethyl alcoholsolution for another 30 minutes. The second (50% ethyl alcohol) solutionwas then replaced for approximately 16 hours by a third solutioncontaining 20% glycerol, 5% weight by volume polyethylene glycol (MW8,000) and 50 unit/ml heparin (molecular weight>3 KD). The tissues werecarefully removed from the third solution, excess liquid was allowed todrain from the tissues and the tissues were dipped in a heparin solutionof 250 unit/ml for a few seconds, prior to freezing the tissues at −70°C. The completely frozen tissues were lyophilized to dryness.

The lyophilized bovine or ovine pericardium tissues obtained above had atranslucent appearance and a slight yellowish tint. They were pliableand did not crack or break by physical manipulations. They could berehydrated by immersion in physiological buffered saline forapproximately 5 minutes at ambient temperature. After rehydration, thetissues were indistinguishable in appearance from the native freshtissues.

Human fibroblasts and umbilical cord vein endothelial cells werecultured on the rehydrated pericardium tissues to study theirbiocompatibility. Round discs of the tissues were cut to fit the bottomof the wells of a 24 well culture plate. Plastic rings were placed ontop of the tissues to hold the tissues down and to ensure a good seal atthe edge of the tissues. Cells were seeded on the tissues in normalculture media for one week. At the end of the incubation period, tissueswere recovered and cut into different portions for histology studies.Histological examination of the cross-section of the tissues showed athin layer of endothelial cells adhering to the surface of the tissues.Cells on the tissues were also released by trypsin and counted. Theseresults showed that the rehydrated tissues are not cytotoxic and arebiocompatible for host cells to attach and proliferate. As is known,attachment and proliferation of endothelial cells and other connectivetissue cells on cardiac implants is essential for the long term survivalof the implant.

The integrity of the collagen fibers in the treated tissues was examinedby melting temperature measurements. For these, tissues were heated inphosphate buffered saline from 37° C. until they shrunk. The shrinkagetemperature of the fresh native tissues and of the lyophilized andrehydrated tissues in accordance with the present invention wasapproximately the same, at approximately 63+1° C., indicating that thecollagen fibers remained intact throughout the lyophilization andrehydration process.

(b) Preparation of Lyophilized Sheep Aortic and Pulmonary Roots

Aortic and pulmonary roots of donor sheep were also treated with theaqueous 25% ethyl alcohol, aqueous 50% ethyl alcohol, aqueous 20%glycerol 5% polyethylene glycol, and subsequent heparin solution andlyophilized, as described above for the bovine and ovine pericardium.

The treated roots were rehydrated and implanted as homografts in thedescending aorta of host sheep. Our results show that after 100 days ofimplantation, the valves were competent and the roots do not appeardifferent from the un-implanted native tissues. The hundred-day explantwas free of fibrin deposition and free of host tissue reaction. Theleaflets of the valve appeared intact and indistinguishable from theunimplanted valve by both gross observation and histologicalexamination.

What is claimed is:
 1. A pliable uncrosslinked soft tissue specimenwhich has been prepared in a process comprising the steps of: (1)treating natural soft tissue obtained from a donor with: (a) liquidcompositions of gradually increasing concentrations of a polar organicsolvent or solvents, until the last of said liquid compositions containsat least approximately 25% and at most 75% by volume of said solvent, ormixture of solvents, the balance being water, the solvent being selectedfrom a group consisting of aliphatic alcohols having 1 to 3 carbons andother water miscible polar organic solvents; (b) thereafter with asecond liquid composition of aqueous glycerol or of low molecular weightpolyethylene glycol having a molecular weight less than approximately1000 D, containing the glycerol or the low molecular weight polyethyleneglycol, or mixtures thereof being in a concentration range ofapproximately 10 to 50% by volume, said second liquid composition alsocontaining approximately 3-20% weight by volume polyethylene glycol of amolecular weight in the range of 6,000 D to 15,000 D and approximately 2to 75 units per milliliter heparin of a molecular weight greater thanapproximately 3 KD; (2) thereafter briefly immersing the soft tissue inan aqueous heparin solution, and (3) thereafter freezing the tissue andlyophilizing the tissue to dryness.
 2. The pliable soft tissue specimenin accordance with claim 1 wherein in the process of preparing thespecimen the polar organic solvents are selected from the groupconsisting of methyl alcohol, ethyl alcohol, iso-propyl alcohol,acetonitrile, acetone and methyl ethyl ketone.
 3. The pliable softtissue specimen in accordance with claim 1 wherein in the process ofpreparing the specimen the natural soft tissue obtained from the donoris treated with liquid compositions of gradually increasingconcentrations of a polar organic solvent or solvents, until the last ofsaid liquid compositions contains at least approximately 50% by volumeof said solvent, or mixture of solvents.
 4. The pliable soft tissuespecimen in accordance with claim 3 wherein in the process of preparingthe specimen the polar organic solvent is ethyl alcohol.
 5. The pliablesoft tissue specimen in accordance with claim 1 wherein in the processof preparing the specimen the second liquid composition containsapproximately 18% by volume of glycerol.
 6. The pliable soft tissuespecimen in accordance with claim 1 wherein in the process of preparingthe specimen the natural soft tissue is treated in succession with twoliquid compositions of a polar organic solvent or solvents, the first ofsaid compositions containing approximately 15 to 35% by volume of thesolvent or solvents, the second of said composition containingapproximately 25 to 75% by volume of the solvent or solvents.
 7. Thepliable soft tissue specimen in accordance with claim 1 wherein theprocess of preparing the specimen further comprises the step ofrehydrating the lyophilized tissue specimen.
 8. A pliable uncrosslinkedsoft tissue specimen, for eventual implantation in a mammal to replaceor augment native tissue, which has been prepared in a processcomprising the steps of: (1) treating natural soft mammalian tissueobtained from a donor with: (a) liquid compositions of graduallyincreasing concentrations of an aliphatic alcohol or mixture ofaliphatic alcohols having 1 to 3 carbon atoms, until the last of saidliquid compositions contains at least approximately 25% by volume ofsaid alcohol or mixture of alcohols, the balance being water; (b)thereafter with a second liquid composition of aqueous glycerolcontaining the glycerol in a concentration range of approximately 10 to50% by volume, said second liquid composition also containingapproximately 3-20% weight by volume polyethylene glycol of a molecularweight in the range of 6,000 D to 15,000 D and approximately 2 to 75units per milliliter heparin of a molecular weight greater thanapproximately 3 KD; (2) thereafter briefly immersing the soft tissue inan aqueous heparin solution of approximately 20 to 500 units permilliliter concentration, and (3) thereafter freezing the tissue andlyophilizing the tissue to dryness.
 9. The soft tissue specimen inaccordance with claim 8 wherein in the process of preparing the specimenthe aliphatic alcohol is ethyl alcohol.
 10. The soft tissue specimen inaccordance with claim 9 wherein in the process of preparing the specimenthe natural soft tissue is treated with said compositions containingethyl alcohol, until the last of said compositions contains at leastapproximately 50% by volume ethyl alcohol.
 11. The soft tissue specimenin accordance with claim 10 wherein in the process of preparing thespecimen the concentration of glycerol in the second liquid compositionis approximately 20% by volume.
 12. The soft tissue specimen inaccordance with claim 11 wherein in the process of preparing thespecimen the concentration of polyethylene glycol in the second liquidcomposition is approximately 5% weight by volume and the molecularweight of said polyethylene glycol is approximately 8,000 D.
 13. Thesoft tissue specimen in accordance with claim 12 wherein in the processof preparing the specimen the natural soft tissue is treated insuccession with two liquid compositions of ethyl alcohol, the first ofsaid compositions containing approximately 15 to 35% by volume of ethylalcohol, the second of said composition containing approximately 25 to75% by volume of ethyl alcohol.
 14. The soft tissue specimen inaccordance with claim 8 wherein the process of preparing the specimenfurther comprises the step of rehydrating the lyophilized tissuespecimen.
 15. The soft tissue specimen in accordance with claim 12wherein in the process of preparing the specimen the natural softmammalian tissue is selected from the group consisting of pericardium,pleura, peritoneum from aortic valve, pulmonary valve, mitral valve,tricuspid valve, tendon and skin.
 16. A pliable soft tissue specimenwhich has been prepared in a process comprising the steps of: (1)treating natural soft tissue obtained from a donor with: (a) liquidcompositions of gradually increasing concentrations of a polar organicsolvent or solvents, until the last of said liquid compositions containsat least approximately 25% and at most 75% by volume of said solvent, ormixture of solvents, the balance being water, the solvent being selectedfrom a group consisting of aliphatic alcohols having 1 to 3 carbons andother water miscible polar organic solvents; (b) thereafter with asecond liquid composition of aqueous glycerol or of low molecular weightpolyethylene glycol having a molecular weight less than approximately1000 D, containing the glycerol or the low molecular weight polyethyleneglycol, or mixtures thereof being in a concentration range ofapproximately 10 to 50% by volume, said second liquid composition alsocontaining approximately 3-20% weight by volume polyethylene glycol of amolecular weight in the range of 6,000 D to 15,000 D and approximately 2to 75 units per milliliter heparin of a molecular weight greater thanapproximately 3 KD; (2) thereafter briefly immersing the soft tissue inan aqueous heparin solution, and (3) thereafter freezing the tissue andlyophilizing the tissue to dryness.
 17. The pliable soft tissue specimenin accordance with claim 16 wherein in the process of preparing thespecimen the polar organic solvents are selected from the groupconsisting of methyl alcohol, ethyl alcohol, iso-propyl alcohol,acetonitrile, acetone and methyl ethyl ketone.
 18. The pliable softtissue specimen in accordance with claim 16 wherein in the process ofpreparing the specimen the natural soft tissue obtained from the donoris treated with liquid compositions of gradually increasingconcentrations of a polar organic solvent or solvents, until the last ofsaid liquid compositions contains at least approximately 50% by volumeof said solvent, or mixture of solvents.
 19. The pliable soft tissuespecimen in accordance with claim 18 wherein in the process of preparingthe specimen the polar organic solvent is ethyl alcohol.
 20. The pliablesoft tissue specimen in accordance with claim 16 wherein in the processof preparing the specimen the second liquid composition containsapproximately 18% by volume of glycerol.
 21. The pliable soft tissuespecimen in accordance with claim 16 wherein in the process of preparingthe specimen the natural soft tissue is treated in succession with twoliquid compositions of a polar organic solvent or solvents, the first ofsaid compositions containing approximately 15 to 35% by volume of thesolvent or solvents, the second of said composition containingapproximately 25 to 75% by volume of the solvent or solvents.
 22. Thepliable soft tissue specimen in accordance with claim 16 wherein theprocess of preparing the specimen further comprises the step ofrehydrating the lyophilized tissue specimen.
 23. A pliable soft tissuespecimen, for eventual implantation in a mammal to replace or augmentnative tissue, which has been prepared in a process comprising the stepsof: (1) treating natural soft mammalian tissue obtained from a donorwith: (a) liquid compositions of gradually increasing concentrations ofan aliphatic alcohol or mixture of aliphatic alcohols having 1 to 3carbon atoms, until the last of said liquid compositions contains atleast approximately 25% by volume of said alcohol or mixture ofalcohols, the balance being water; (b) thereafter with a second liquidcomposition of aqueous glycerol containing the glycerol in aconcentration range of approximately 10 to 50% by volume, said secondliquid composition also containing approximately 3-20% weight by volumepolyethylene glycol of a molecular weight in the range of 6,000 D to15,000 D and approximately 2 to 75 units per milliliter heparin of amolecular weight greater than approximately 3 KD; (2) thereafter brieflyimmersing the soft tissue in an aqueous heparin solution ofapproximately 20 to 500 unit per milliliter concentration, and (3)thereafter freezing the tissue and lyophilizing the tissue to dryness.24. The soft tissue specimen in accordance with claim 23 wherein in theprocess of preparing the specimen the aliphatic alcohol is ethylalcohol.
 25. The soft tissue specimen in accordance with claim 24wherein in the process of preparing the specimen the natural soft tissueis treated with said compositions containing ethyl alcohol, until thelast of said compositions contains at least approximately 50% by volumeethyl alcohol.
 26. The soft tissue specimen in accordance with claim 25wherein in the process of preparing the specimen the concentration ofglycerol in the second liquid composition is approximately 20% byvolume.
 27. The soft tissue specimen in accordance with claim 26 whereinin the process of preparing the specimen the concentration ofpolyethylene glycol in the second liquid composition is approximately 5%weight by volume and the molecular weight of said polyethylene glycol isapproximately 8,000 D.
 28. The soft tissue specimen in accordance withclaim 27 wherein in the process of preparing the specimen the naturalsoft tissue is treated in succession with two liquid compositions ofethyl alcohol, the first of said compositions containing approximately15 to 35% by volume of ethyl alcohol, the second of said compositioncontaining approximately 25 to 75% by volume of ethyl alcohol.
 29. Thesoft tissue specimen in accordance with claim 23 wherein the process ofpreparing the specimen further comprises the step of rehydrating thelyophilized tissue specimen.
 30. The soft tissue specimen in accordancewith claim 23 wherein in the process of preparing the specimen thenatural soft mammalian tissue is selected from the group consisting ofpericardium, pleura, peritonium from aortic valve, peritonium frompulmonary valve, peritonium from mitral valve, peritonium from tricuspidvalve, tendon and skin.